Mouse Antibody Cloning

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Mouse Light Chain

[edit]

Alignment

[edit]
K       --ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ	58
L1      GQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSK	60
L2      GQPKSTPTLTVFPPSSEELKENKATLVCLISNFSPSGVTVAWKANGTPITQGVDTSNPTK	60
           .::*::::******:*  . *::** :.:* *  :.* ** :*:   ;:*: .:   :

K       DSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC-	106
L1      Q-SNNKYMASSYLTLTARAWERHSSYSCQVTHEGHT--VEKSLSRADCS	106
L2      E--GNKFMASSFLHLTSDQWRSHNSFTCQVTHEGDT--VEKSLSPAECL	105
        :  ...:  ** * **   :. *.*::*:.**:  *  : **:.  :* 

L1      GQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSK	60
L2      GQPKSTPTLTVFPPSSEELKENKATLVCLISNFSPSGVTVAWKANGTPITQGVDTSNPTK	60
        *****:*::*:********: ******* *::* *. *** **.:***:***::*::*:*

L1      QSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS	106
L2      E-GNKFMASSFLHLTSDQWRSHNSFTCQVTHEGDTVEKSLSPAECL	105
        : .**:****:* **:  *. *.*::*******.******* *:*

[edit]

Oligo for RT: Light chain (kappa & lambda)

[edit]
t(g/c)ctcactggatggtgggaa
tSctcactggatggtgggaa
{ttcccaccatccagtgag (c/g)a}
  F  P  P  S  S  E  Q

Kappa

[edit]

constant region

GCAGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG

ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDS KDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 

gcagatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctgga
 A  D  A  A  P  T  V  S  I  F  P  P  S  S  E  Q  L  T  S  G 
ggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtgg
 G  A  S  V  V  C  F  L  N  N  F  Y  P  K  D  I  N  V  K  W 
aagattgatggcagtgaacgacaaaatggcgtcctgaacagttggactgatcaggacagc
 K  I  D  G  S  E  R  Q  N  G  V  L  N  S  W  T  D  Q  D  S 
aaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacga
 K  D  S  T  Y  S  M  S  S  T  L  T  L  T  K  D  E  Y  E  R 
cataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagc
 H  N  S  Y  T  C  E  A  T  H  K  T  S  T  S  P  I  V  K  S 
ttcaacaggaatgagtgttag
 F  N  R  N  E  C  -

[edit]
aligment of some kappa light chain VL domain
4      elvltqspttmaaspgekititcsasssissnylhwyqqkpgfspklliyrtsnlasgvp
2      qivltqspaimsaslgdrvtmtctasssvsssylhwyqqkpgsspklwiystsnlasgvp
1      envltqspaimsaspgekvtmacrasssvsstylhwyqqksgaspklliystsnlasgvp
3      envltqspaimsaspgekvtmtcrasssvsssylhwyqqksgaspklwiystsnlasgvp
5      -ivmtqtqkfmstsvgervsitckasqnvgt-nvawyqqkggqslelliygasnrhtgvp
6      dvvmtqthkfmstsvgdrvsitckasqdvsg-avawyqqksgqspkllismasqrytgvp
         *:**:   *::* *::::::* **..:.   : ***** * * :* *  :*:  :***

4      arfsgsgsgtsysltigtmeaedvatyycqqgssipftfgsgtkleikr
2      arfsgsgsgtsysltissmeaedaatyychqfhr-sltfgsgtkleikr
1      arfsgsgsgtsysltissveaedaatyycqqysgypltfgagtklelkr
3      arfsgsgsgtsysltissveaedaatyycqqysgypytfgggtkleikr
5      drftgrgsgtdftltitnvqsedmtnyfceqysrypltfgagtklelkr
6      drftgsgsgtdftftissvqaedlavyycqqhyaipltfgagtklelkr
        **:* ****.:::** .:::** : *:*.*      ***.*****:**

Lambda 1

[edit]

GGCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCCACCTTCCTCTGAAGAGCTCGAGACTAACAAGGCCACACTGGTGTGTACGATCACTGATTTCTACCCAGGTGTGGTGACAGTGGACTGGAAGGTAGATGGTACCCCTGTCACTCAGGGTATGGAGACAACCCAGCCTTCCAAACAGAGCAACAACAAGTACATGGCTAGCAGCTACCTGACCCTGACAGCAAGAGCATGGGAAAGGCATAGCAGTTACAGCTGCCAGGTCACTCATGAAGGTCACACTGTGGAGAAGAGTTTGTCCCGTGCTGACTGTTCCTAG

GQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS 

ggccagcccaagtcttcgccatcagtcaccctgtttccaccttcctctgaagagctcgag
 G  Q  P  K  S  S  P  S  V  T  L  F  P  P  S  S  E  E  L  E 
actaacaaggccacactggtgtgtacgatcactgatttctacccaggtgtggtgacagtg
 T  N  K  A  T  L  V  C  T  I  T  D  F  Y  P  G  V  V  T  V 
gactggaaggtagatggtacccctgtcactcagggtatggagacaacccagccttccaaa
 D  W  K  V  D  G  T  P  V  T  Q  G  M  E  T  T  Q  P  S  K 
cagagcaacaacaagtacatggctagcagctacctgaccctgacagcaagagcatgggaa
 Q  S  N  N  K  Y  M  A  S  S  Y  L  T  L  T  A  R  A  W  E 
aggcatagcagttacagctgccaggtcactcatgaaggtcacactgtggagaagagtttg
 R  H  S  S  Y  S  C  Q  V  T  H  E  G  H  T  V  E  K  S  L 
tcccgtgctgactgttcctag
 S  R  A  D  C  S  -

Lambda 2

[edit]

GGTCAGCCCAAGTCCACTCCCACTCTCACCGTGTTTCCACCTTCCTCTGAGGAGCTCAAGGAAAACAAAGCCACACTGGTGTGTCTGATTTCCAACTTTTCCCCGAGTGGTGTGACAGTGGCCTGGAAGGCAAATGGTACACCTATCACCCAGGGTGTGGACACTTCAAATCCCACCAAAGAGGGCAACAAGTTCATGGCCAGCAGCTTCCTACATTTGACATCGGACCAGTGGAGATCTCACAACAGTTTTACCTGCCAAGTTACACATGAAGGGGACACTGTGGAGAAGAGTCTGTCTCCTGCAGAATGTCTCTAA

GQPKSTPTLTVFPPSSEELKENKATLVCLISNFSPSGVTVAWKANGTPITQGVDTSNPTKEGNKFMASSFLHLTSDQWRSHNSFTCQVTHEGDTVEKSLSPAECL 


ggtcagcccaagtccactcccactctcaccgtgtttccaccttcctctgaggagctcaag
 G  Q  P  K  S  T  P  T  L  T  V  F  P  P  S  S  E  E  L  K 
gaaaacaaagccacactggtgtgtctgatttccaacttttccccgagtggtgtgacagtg
 E  N  K  A  T  L  V  C  L  I  S  N  F  S  P  S  G  V  T  V 
gcctggaaggcaaatggtacacctatcacccagggtgtggacacttcaaatcccaccaaa
 A  W  K  A  N  G  T  P  I  T  Q  G  V  D  T  S  N  P  T  K 
gagggcaacaagttcatggccagcagcttcctacatttgacatcggaccagtggagatct
 E  G  N  K  F  M  A  S  S  F  L  H  L  T  S  D  Q  W  R  S 
cacaacagttttacctgccaagttacacatgaaggggacactgtggagaagagtctgtct
 H  N  S  F  T  C  Q  V  T  H  E  G  D  T  V  E  K  S  L  S 
cctgcagaatgtctctaa
 P  A  E  C  L  -

Alignment 

Lambda1      GQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSK
lambda2      GQPKSTPTLTVFPPSSEELKENKATLVCLISNFSPSGVTVAWKANGTPITQGVDTSNPTK
             *****:*::*:********: ******* *::* *. *** **.:***:***::*::*:*

Lambda1      QSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS
lambda2      E-GNKFMASSFLHLTSDQWRSHNSFTCQVTHEGDTVEKSLSPAECL
             : .**:****:* **:  *. *.*::*******.******* *:*

Heavy Chain constant region

[edit]
gccaaaacaacagccccatcggtctatccactggcccctgtgtgtggagatacaactggc
 A  K  T  T  A  P  S  V  Y  P  L  A  P  V  C  G  D  T  T  G 
tcctcggtgactctaggatgcctggtcaagggttatttccctgagccagtgaccttgacc
 S  S  V  T  L  G  C  L  V  K  G  Y  F  P  E  P  V  T  L  T 
tggaactctggatccctgtccagtggtgtgcacaccttcccagctgtcctgcagtctgac
 W  N  S  G  S  L  S  S  G  V  H  T  F  P  A  V  L  Q  S  D 
ctctacaccctcagcagctcagtgactgtaacctcgagcacctggcccagccagtccatc
 L  Y  T  L  S  S  S  V  T  V  T  S  S  T  W  P  S  Q  S  I 
acctgcaatgtggcccacccggcaagcagcaccaaggtggacaagaaaatt
 T  C  N  V  A  H  P  A  S  S  T  K  V  D  K  K  I 

ctcagggaaataacccttgaccaggcatcctagagtcac
(gtgactctaggatgcctggtcaagggttatttccctgag) 
  V  T  L  G  C  L  V  K  G  Y  F  P  E

Primer for RT
Mouse-κγLC-RT tSctcactggatggtgggaa
Mouse-IgG-HC-RT ctcagggaaataacccttgac
CTC AGG GAA ATA ACC CTT GAC

Alignment

[edit]
G1       AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSD	60
G2a      AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSD	60
G2b      AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSG	60
         **** ******** ..  *.* ************* **:*********.******:***.

G1       LYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDC-----GCKPC----ICTV	111
G2a      LYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGP--TIKPCPPC----KCPA	114
G2b      LYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPA	120
         ***:****** **  **:::**.********.****: * .       * **     * .

G1       P---EVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQP	168
G2a      PNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQT	174
G2b      PNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVRISWFVNNVEVHTAQTQT	180
         *      *******: **** *:*:* *******:*:***:*::****::********* 

G1       REEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTI	228
G2a      HREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVL	234
G2b      HREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYIL	240
         :.*::***:* ** *** ****:.******:**.  :*:***:**** **  :***** :

G1       PPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLN	288
G2a      PPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLR	294
G2b      PPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLD	300
         *** *::::..*:***::..* * ** ***  **:   ***:* *:::::****:**** 

G1       VQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK	324
G2a      VEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK	330
G2b      IKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK	336
         ::..:*   ::::*.* ****:*::  *::*::***


IgG1

[edit]

GCTAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCGCAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA

AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK 

alignment of some IgG1 VH domain

6      -----------------------------qvqlkqsgpglvapsqslsitctvsgfslt-
7      matgsrtslllafgl---lclpwlqegsaqvtlkesgpgilrpsqtldltcsfsgfslst
2      ----------mnfglsliflvlilkgvqcevklvesggglvkpggslklscaasgftfs-
1      -----------------------------qvqlqqsgaelvrpgssvkisckasgyafs-
5      matgsrtslllafgl---lclpwlqegsaqvqlqqsgaelvkpgssvkisckasgytft-
3      ----------mgwswiflfllsgtagvlsevqlqqsgpervkpgasvkisckasgytft-
4      ----------mgwswiflfllsgtagvlsevqlqqsgpervkpgasvkisckasgytft-
8      ----------mgwtwifililsvttgvhsdvqlqqsgpelekpgasvkisckasgfslp-
                                    :* * :**     *. ::.::*  **:::  

6      -gygvnwvrqspgkglewlgmiwg-dgrtdyksalksrlsitkdnsksqvflkmnslqtd
7      sglsvgwirqpsgkglewlahiww-ddvkyfnpslksrltiskdssrnqvflkitsvdta
2      -sygmswvrqtpekrlewvatisgggshtyypdivkgrftisrdnaknnlylqmsslrse
1      -sywmnwvkqrpgqglewigqiypgdsdtnyngkfkgkatltadkssstaymqlssltse
5      -sddmhwikqqpgnglewigwiypgnddtkynqkfngkatltadkssstvymqlssltse
3      -dyymnwvkqshgkslewigdinpnnggtsynqkfegkatltvdkssstaymelrsltse
4      -dyymnwvkqshgkslewigdinpnnggtsynqkfegkatltvdkssstaymelrsltse
8      -ghninwivqrngkslewignidpyyggtnfnpkfkgkatltvdkssstlymhltslqse
        .  : *: *   : ***:. *    . . :   .:.: ::: *.: .  ::.: *: : 

6      dtaryfcasdyyg-sgsfaywgqgtlvtvsa
7      dsatyhctrgplg-hg-fdywgqgtlvtvsa
2      dtalyhcarrdwd-gyyfdywgqgttltvss
1      dsavyfcardyg--kdhfdywgqgttltvss
5      dsavyfcargdlnyggsmdawgqgtsvtvss
3      dsavyycarg--g-lhwfpywgqgtlvtvsa
4      dsavyycarg--g-lhwfpywgqgtlvtvsa
8      dsavyycarrrdg-nygftywgqgtlvtvsa
       *:* *.*:         :  ***** :***:

Cloning site:
Nhe1
GCTAGCAGC GCTAAAACGAC
 A  S  S   A  K  T

IgG2a

[edit]

GCTAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAATGA

AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

IgG2b

[edit]

GCTAAAACAACACCCCCATCAGTCTATCCACTGGCCCCTGGGTGTGGAGATACAACTGGTTCCTCTGTGACTCTGGGATGCCTGGTCAAGGGCTACTTCCCTGAGTCAGTGACTGTGACTTGGAACTCTGGATCCCTGTCCAGCAGTGTGCACACCTTCCCAGCTCTCCTGCAGTCTGGACTCTACACTATGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCAAGTCAGACCGTCACCTGCAGCGTTGCTCACCCAGCCAGCAGCACCACGGTGGACAAAAAACTTGAGCCCAGCGGGCCCATTTCAACAATCAACCCCTGTCCTCCATGCAAGGAGTGTCACAAATGCCCAGCTCCTAACCTCGAGGGTGGACCATCCGTCTTCATCTTCCCTCCAAATATCAAGGATGTACTCATGATCTCCCTGACACCCAAGGTCACGTGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGACGTCCGGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTATCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCATCACCCATCGAGAGAACCATCTCAAAAATTAAAGGGCTAGTCAGAGCTCCACAAGTATACATCTTGCCGCCACCAGCAGAGCAGTTGTCCAGGAAAGATGTCAGTCTCACTTGCCTGGTCGTGGGCTTCAACCCTGGAGACATCAGTGTGGAGTGGACCAGCAATGGGCATACTGAGGAGAACTACAAGGACACCGCACCAGTCCTGGACTCTGACGGTTCTTACTTCATATACAGCAAGCTCGATATAAAAACAAGCAAGTGGGAGAAAACAGATTCCTTCTCATGCAACGTGAGACACGAGGGTCTGAAAAATTACTACCTGAAGAAGACCATCTCCCGGTCTCCGGGTAAATGA

AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVRISWFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK 

IgG1       AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSD
IgG2a      AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSD
IgG2b      AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSG
           **** ******** ..  *.* ************* **:*********.******:***.

IgG1       LYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDC-----GCKPC----ICTV
IgG2a      LYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGP--TIKPCPPC----KCPA
IgG2b      LYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPA
           ***:****** **  **:::**.********.****: * .       * **     * .

IgG1       P---EVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQP
IgG2a      PNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQT
IgG2b      PNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVRISWFVNNVEVHTAQTQT
           *      *******: **** *:*:* *******:*:***:*::****::********* 

IgG1       REEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTI
IgG2a      HREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVL
IgG2b      HREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYIL
           :.*::***:* ** *** ****:.******:**.  :*:***:**** **  :***** :

IgG1       PPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLN
IgG2a      PPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLR
IgG2b      PPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLD
           *** *::::..*:***::..* * ** ***  **:   ***:* *:::::****:**** 

IgG1       VQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK
IgG2a      VEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK
IgG2b      IKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK
           ::..:*   ::::*.* ****:*::  *::*::***

IgG2a VH domain alignment

[edit]
CBI70317     mtslfslqllstqdlamgwsciilflvaaagrvhsqvqlqqpgaelvkpgasvkmsckas
ADA84145     ----------------mavlvlflclvafpscvlsqvqlkesgpglvapsqslsitctvs
AGH20709     --------------------------mdwtwrvlcqitlkesgpgilqpsqtlsltcsfs
CAD18854     ----------------mgrltssflllivpayvlsqvtlkesgpgilqpsqtlsltcsfs
AAI08376     ----------------mylglscvfivfllkgvqcevkldesggglvqpgrsmklscvas


CBI70317     gysftsywm--nwvkqrpgrglewigridpsd-n-ethynqdfkdkvtltvdkssstvyi
ADA84145     gfsltnygv--hwvrqppgkglewlgmiwagg---stnynsalmsrlninkdnsksqvfl
AGH20709     gfslttygigagwirqppgkglewlthiwwnd---nkyyntalrsrltiskdssnnqvll
CAD18854     gfslstygmgvgwirqpsgkglewlaniwwnd---hkyynsalksrltiskdtsnnqvfl
AAI08376     gftfsdywm--nwvhqspekglewvaqirnkpynyvtyysdsvrgrftisrddskssvyl


CBI70317     qlssltsedsavyycgr----lgyv-ygfdywgqgttltvssakttaps
ADA84145     emnslqtddtalyfcareat-sgyvdyavdywgqgtsvtvssakttaps
AGH20709     kianvdtadtatyycl----------ygytywgqgtlvtvsaakttaps
CAD18854     kissvdtadtatyycah----ysnyayaldywgqgtsvtvssakttaps
AAI08376     qmnnlraedtgiyyctssntyysyedy-fdywgqgttitvssakttaps

IgG2b VH domain alignment

[edit]
7       mdrltssflllivpayvlsqvtlkesgpgilkpsqtlsltcsfsgfslstsgmgvgwirq
1       mdwlwnllflmaaaqsaqaqiqlaqsgpelkepgetvkiscktsgftfityg--vswvkq
5       mkcswvifflmavvigvnsevqllqsgtelvrpgasvklsctasgfsikdsl--lhwvmq
4       mewpcillfllsvtegvhsqvqllqsgpelvkpgasvkiscrasgyafsksw--mnwvkr
9       mewiwiflfilsgtagvqsqvqlqqsgaelarpgasvklsckasgytltsyg--iswvkq
10      mewiwiflfilsgtagvryqvqlqqsgaelarpgasvklsckasgytltsyg--iswvkq
3       mgwscimlflaatatgvhsqvqlqqpgaelvmpgasvrlsckasgytftryw--mhwlkq
8       mewswiflfllsgtagvhsevqlqqsgpelvnpgasvkmsckasgytfityv--mhwvkq
2       merhwiflfllsvtagvhsqvqlqqsgaelakpgasvkmsckasgytftsyw--mhwvkq
6       merhwiflflfsvtagvhsqvqlqqsgaelakpgasvkmsckasgysftsyw--mhwvkq
        *      :::      .  :: * : *  :  *. :: ::*  **:::      : *: :

7       psgkglewlahiww-dddkhynpslksqltiskdssrnqvflkitsvdtadtatyycvrr
1       apgkglkwmgwintysglptyapdfkgrfafslktsastaylqinnlknedtatyfcvin
5       rpeqglewigwidpedgetkyvpkfqdkatltaatssntaylqlssltsgdtatyyclfy
4       rpgkglewigrifpgdgdthysgkfqgkakltadkssvtaflqltsltsedsavyfcard
9       rtgqglewigeiypgsgnsyfnekfkgkatltvdkssstaylhlssltsedsavyfcagp
10      rtgqglewigeiypgsgnsyfnekfkgkatltvdkssstaylhlssltsedsavyfcagp
3       rpgrglewigwidpnsdgtkynekfkgkatltvdipsstafmqlssltsedsavyycagn
8       kpgqglewigyinpnkdgtkfnekfkgkatltsdkssntaymelssltsedsavyycard
2       rpgqglewigyinpssgyteynqkfkdkatltadkssstaymqlssltsedsavyycars
6       rpgqglewigyinpstgyteynqkfkdkatltadkssstaymqlssltsedsavyycarg
           :**:*:. *    .   :  .::.:  ::       .::.:..: . *:*.*:*   

7       sfsygssrdyfdywgqgttltvssakttpp
1       dpy------amdywgqgtsvtvssakttpp
5       gydd--grdyfdywgqgttitvssakttpp
4       sdy----gdyfddwgqgatvtvssakttpp
9       rqv---gllpfgywgqgtlvtasaakttpp
10      rqv---gllpfgywgqgtlvtasaakttpp
3       fg---------aywgqgtlvtvsaakttpp
8       ydy-----dwfaywgqgtlvtvsaakttpp
2       fyyy--segamdywgqgtsvtvssakttpp
6       dgyy---egwfaywgqgtlvtvsaakttpp
                     ****: :*.*:******

Mouse IgM

[edit]

SQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGKNRDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLV

secreted form TERTVDKSTGKPTLYNVSLIMSDTGGTCY

membrane bound form EGEVNAEEEGFENLWTTASTFIVLFLLSLFYSTTVTLFKVK

Secreted Form from BALB/c AGAGTCAGTCCTTCCCAAATGTCTTCCCCCTCGTCTCCTGCGAGAGCCCCCTGTCTGATAAGAATCTGGTGGCCATGGGCTGCCTAGCCCGGGACTTCCTGCCCAGCACCATTTCCTTCACCTGGAACTACCAGAACAACACTGAAGTCATCCAGGGTATCAGAACCTTCCCAACACTGAGGACAGGGGGCAAGTACCTAGCCACCTCGCAGGTGTTGCTGTCTCCCAAGAGCATCCTTGAAGGTTCAGATGAATACCTGGTATGCAAAATCCACTACGGAGGCAAAAACAGAGATCTGCATGTGCCCATTCCAGCTGTCGCAGAGATGAACCCCAATGTAAATGTGTTCGTCCCACCACGGGATGGCTTCTCTGGCCCTGCACCACGCAAGTCTAAACTCATCTGCGAGGCCACGAACTTCACTCCAAAACCGATCACAGTATCCTGGCTAAAGGATGGGAAGCTCGTGGAATCTGGCTTCACCACAGATCCGGTGACCATCGAGAACAAAGGATCCACACCCCAAACCTACAAGGTCATAAGCACACTTACCATCTCTGAAATCGACTGGCTGAACCTGAATGTGTACACCTGCCGTGTGGATCACAGGGGTCTCACCTTCTTGAAGAACGTGTCCTCCACATGTGCTGCCAGTCCCTCCACAGACATCCTAACCTTCACCATCCCCCCCTCCTTTGCCGACATCTTCCTCAGCAAGTCCGCTAACCTGACCTGTCTGGTCTCAAACCTGGCAACCTATGAAACCCTGAATATCTCCTGGGCTTCTCAAAGTGGTGAACCACTGGAAACCAAAATTAAAATCATGGAAAGCCATCCCAATGGCACCTTCAGTGCTAAGGGTGTGGCTAGTGTTTGTGTGGAAGACTGGAATAACAGGAAGGAATTTGTGTGTACTGTGACTCACAGGGATCTGCCTTCACCACAGAAGAAATTCATCTCAAAACCCAATGAGGTGCACAAACATCCACCTGCTGTGTACCTGCTGCCACCAGCTCGTGAGCAACTGAACCTGAGGGAGTCAGCCACAGTCACCTGCTTGGTGAAGGGCTTCTCTCCTGCAGACATCAGTGTGCAGTGGCTTCAGAGAGGGCAACTCTTGCCCCAAGAGAAGTATGTGACCAGTGCCCCGATGCCAGAGCCTGGGGCCCCAGGCTTCTACTTTACCCACAGCATCCTGACTGTGACAGAGGAGGAATGGAACTCCGGAGAGACCTATACCTGTGTTGTAGGCCACGAGGCCCTGCCACACCTGGTGACCGAGAGGACCGTGGACAAGTCCACTGGTAAACCCACACTGTACAATGTCTCCCTGATCATGTC TGACACAGGCGGCACCTGCTATTGA

Reverse Transcriptase Gene Specific Primers

[edit]

Cloning from Hybridoma Murine SP2/0-Ag14 (SP2/0)

Mouse-IgM-RT gacgagggggaagacatttgggaa  Published primers (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4278175/ 

Igκ, AGTGGCCTCACAGGTAT
(ATACCTGTGAGGCCACT, TCEAT)

IgM, GGTATTCATCTGAACCTTCAAG
(CTTGAAGGTTCAGATGAATACC, LEGSDEY)

IgG, ACAGTCACTGAGCTGC
(GCAGCTCAGTGACTGT, SSSVTV)

https://www.sciencedirect.com/science/article/pii/S0161589017304789#tbl0005

IgG1_2a_2b-CH1-RT, CTGGACAGGGATCCAGAGTTCCA 
tggaactctggatccctgtccag
 W  N  S  G  S  L  S 

IgG1_Hinge-RT, GGCTTACAACCACAATCCCTG 
cagggattgtggttgtaagcc
  R  D  C  G  C  K  

LC-kappa-RT, GGATACAGTTGGTGCAGCATC 
gatgctgcaccaactgtatcc
 D  A  A  P  T  V  S 

IgG1_2b-CH1-RT GACAGATGGGGGTGTCGTTTTGGC
gccaaaacgacacccccatctgtc
 A  K  T  T  P  P  S  V
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